TWO STEP PROTOCOL FOR ALZHEIMER'S SOLUBLE AMYLOID BETA PROTEIN ISOLATION FROM NORMAL HUMAN CEREBROSPINAL FLUID LIPOPROTEIN
A.R. Koudinov1,2*, T.T. Berezov1, A. Kumar3, R. Beavis3 and N.V. Koudinova1,2
1. Russian Academy of Medical Sciences, National Mental Health Research Center, Institute of Biomedical Chemistry, Timoshenko 38-27, Moscow 121359, Russian Federation
2. Department of Neurobioliology, Weizmann Institute, Rehovot 76100, Israel
3. Department of Pathology and Pharmacology, New York University Medical Center, 560 First Avenue, New York, NY 10016, USA
In normal human plasma and CSF the soluble amyloid beta protein (sAb) is associated with the high density lipoprotein (HDL) (Biochem Biophys Res Commun (1994) 205, 1164-1171). The aim of the present work was to develop simple protocol for isolation of sAb from the CSF-HDL. To this end HDL were first obtained by 60 h ultracentrifugation (UC) of the CSF at the density of 1.25 g/ml (39 g KBr per 100 ml of the CSF) and the sAb and apolipoproteins (apo) were fractionated by reverse phase (RP) HPLC on a C4 analytical column over the 30 min linear gradient of 20 to 80% acetonitrile in 0.1% TFA. The sAb and apo fractions obtained were characterized by SDS/PAGE, immunoblot and mass spectrometry. The following 60-min-RP-HPLC step yielded additional ~2 fold purification of the sAb. Once calibrated with anti-sAb antibodies (6E10, Senetek, PLC), the method (TABLE) can be used for the routine preparation of the native CSF sAb with no need for expensive antibodies.
TABLE. Purification of sAb from normal human CSF
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